Project Summary Adoptive cell therapy (ACT) is an emerging immunotherapy which shows significant promise in treating both leukemia and solid tumors, as well as certain infectious diseases. ACT comprises the isolation and ex vivo expansion of cytotoxic T lymphocytes (CTLs) recognizing epitopes of a mutated or aberrantly expressed protein present almost exclusively on the surface of a patient?s tumor cells. To meet the need for personalized enrichment of the most effective CTLs, this Phase II STTR will employ Cell Microsystems? proprietary CellRaft technology to develop a high-throughput, automated platform, the CLEAR AIR? System, to efficiently screen for and isolate highly active, tumor-directed CTLs on a single cell basis. The CellRaft? technology is based on a unique microwell array recently developed by the Allbritton Lab at the University of North Carolina at Chapel Hill. Cell Microsystems has an exclusive license to the CellRaft? technology, currently marketing both the consumable CellRaft Arrays as well as microscope accessories enabling imaging, isolation and retrieval of single cells from the array. In Phase I we used the CellRaft technology to assay the cytotoxic activity and kinetics of CTLs. T-cells were pre-treated with peptide epitopes known to be expressed on influenza-infected cells. Epitope-treated and untreated CTL populations were then co-incubated with cells expressing the influenza epitopes and monitored for cytotoxic activity. By performing these assays at the single cell level, a unique population of ?super killers? were identified as those exhibiting cytotoxicity at least 4 standard deviations above mean cytotoxicity levels. These super killers were enriched by approximately 6-fold in epitope treated CTLs compared to untreated CTLs. These data indicate that the CellRaft technology is amenable to performing CTL cytotoxicity assays and retrieval of CTLs for downstream propagation and analysis. In Phase II we will build on these data by: 1) designing and testing a 96-well CellRaft Array containing 96 small arrays within a single consumable to test multiple conditions and immunoreactive epitopes in the cytotoxicity assay; 2) using this new 96-well CellRaft array to assay sequence dependent cytotoxicity of tumor cell-expressed epitopes; 3) enabling rapid imaging and long-term culture on the automated CLEAR AIR? System for longitudinal studies such as these and 4) performing external validation studies at both UNC Chapel Hill and MD Anderson Cancer Center for novel CTL recognition proteins. This workflow will allow analysis of the T-cell receptor without requiring the best killing cells to be propagation-capable, an issue observed in Phase I with the best killing cells. Commercially, we view the CellRaft System, as both a research tool enabling correlation of killing efficiency with the molecular characteristics of the T cell receptor (TCR) and eventually as a clinical tool for identifying efficacious TCR sequences as well as other time course-based phenotypic screens.